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Strongyloidiasis, caused by Strongyloides stercoralis, is a neglected parasitic disease that represents a serious public health problem. In immunocompromised patients, this parasitosis can result in hyperinfection or disseminated disease with high levels of mortality. In previous studies, the mRNAs encoding for the 14-3-3 and major antigen proteins were found to be expressed at high levels in S. stercoralis L3 larvae, suggesting potential key roles in parasite-host interactions. We have produced them as recombinant proteins (rSs14-3-3 and rSsMA) in a bacterial protein expression system. The serum levels of anti-rSs14-3-3 and anti-rSsMA IgGs are increased upon infection with S. venezuelensis, validating the use of the mouse model since the native 14-3-3 and MA proteins induce an immune response. Each recombinant protein was formulated in the adjuvant adaptation (ADAD) vaccination system and injected twice, subcutaneously, in CD1 mice that were experimentally infected with 3000 S. venezuelensis L3 to evaluate their protective and immunomodulatory activity. Our results, including the number of parthenogenetic females, number of eggs in stool samples and the analysis of the splenic and intestinal indexes, show that the vaccines did not protect against infection. The immunization with rSs14-3-3 induced changes in the cytokine profile in mice, producing higher expression of IL-10, TGF-β, IL-13 and TNF-α in the spleen, suggesting a Th2/Treg-type response with an increase in TNF-α levels, confirming its role as an immunomodulator.

Background: Schistosomiasis has been identified as a major public health problem in tropical countries. The present study aimed to investigate the schistosomicidal effects of the methanolic extract of Argemone mexicana L. and its active component, berberine against Schistosoma mansoni on in-vitro experiments. Methods: S. mansoni adults were used. Various concentrations of the methanolic extract (10 - 200 µg/ml) and berberine (2.5 - 50 µM) were tested from 24 to 72 h. The viability of S. mansoni was confirmed with an invertoscope-microscope. Furthermore, cytotoxic (Hemolysis test), and antioxidant (DPPH radical scavenging assay) capacities were determined. Results: The viability tests on S. mansoni showed that A. mexicana at 50 μg/mL is lethal at 48 h and berberine at 10 μM is lethal at 24 h. The hemolytic activity at 1,000 μg/mL was 2.9% for A. mexicana and 90.2% for berberine. The antioxidant capacities shown by A. mexicana and berberine, were EC50 156.3 and 84.1 μg/mL, respectively. Conclusion: The extract of A. mexicana and berberine demonstrated high antischistosomal activities in low concentration and short exposure time on the in-vitro model.

High levels of infection and severe liver fibrosis in schistosomiasis appear only in a few cases of infected people with high susceptibility. Tissue damage is caused by the inflammatory response to eggs trapped in the liver. The genetic background influences susceptibility to schistosome infection. To assess the genetic basis of susceptibility to schistosomiasis and identify the chromosomic regions involved, we used a backcross strategy to generate a mouse cohort with high variation in schistosomiasis susceptibility. Thus, we crossed the resistant C57BL/6 mouse strain with the susceptible CBA one; and later; the F1 females (C57 x CBA) were backcrossed with CBA males generating the F1BX cohort. The spectrum of phenotypes in the F1BX mice showed gradation from mild to severe disease, lacking a fully resistant group. We differentiated four levels of infection intensity using cluster and principal component analyses and K-means based on parasitological, pathological and immunological trait measurements. Mice were massively genotyped with 961 informative SNPs. We identify 19 new quantitative trait loci (QTL) associated with phenotype indicators of parasite burden, liver lesion, white blood cell populations, and antibody responses evaluated in the backcross cohort. Two QTLs on chromosomes 15 and 18 were simultaneously linked to the number of granulomas, grade of liver lesion and IgM levels. The human syntenic regions are located in chromosomes 8 and 18. None of the significant QTL identified coincided with previously reported mice association or were syntenic with human chromosomes.Author summaryHigh number of cases of infection and high levels of infection and liver fibrosis in schistosomiasis appear only in a few cases of people with high susceptibility, because the genetic background influences this susceptibility. To assess this, we used a backcross strategy crossing a resistant strain of mouse C57BL/6 with another susceptible CBA, and later F1 females were backcrossed with males susceptible, generating the F1BX cohort, which showed gradation from mild to severe disease. We differentiated four levels of infection according to cluster, principal component analysis and K-means, and based on parasitological, pathological and immunological measurements. Mice were massively genotyped and 19 quantitative trait loci (QTL) were identified, associated with phenotype indicators evaluated in backcross cohort. Two QTLs on chromosomes 15 and 18 were simultaneously linked to the number of granulomas, grade of liver lesion and IgM levels. None of the significant QTL identified coincided with previously reported mice association or were syntenic with human chromosomes.

Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

Schistosoma mansoni is less susceptible to the antiparasitic drug ivermectin than other helminths. By inhibiting the P-glycoprotein or cytochrome P450 3A in mice host or parasites in a murine model, we aimed at increasing the sensitivity of S. mansoni to the drug and thus preventing infection. We assigned 124 BALB/c mice to no treatment, treatment with ivermectin only or a combination of ivermectin with either cobicistat or elacridar once daily for three days before infecting them with 150 S. mansoni cercariae each. The assignment was done by batches without an explicit randomization code. Toxicity was monitored. At eight weeks post-infection, mice were euthanized. We determined number of eggs in intestine and liver, adult worms in portal and mesenteric veins. Disease was assessed by counting granulomas/cm2 of liver and studying organ weight indices and total weight. IgG levels in serum were also considered. No difference between groups treated with ivermectin only or in combination with cobicistat or elacridar compared with untreated, infected controls. Most mice treated with ivermectin and elacridar suffered severe neurological toxicity. In conclusion, systemic treatment with ivermectin, even in the presence of pharmacological inhibition of P-glycoprotein or cytochrome P450 3A, did not result in effective prophylaxis for S. mansoni infection in an experimental murine model.

Schistosomiasis is a parasitic disease that affects 143 million people in endemic countries. This work analyzed overexpressed sequences from the cercaria phase to the early schistosomulum phase using bioinformatics tools to predict host interaction and selected proteins for predicting T cell epitopes. The final peptides were chemically synthesized, and their toxicity was evaluated in vitro. Peptides were formulated in the Adjuvant Adaptation (ADAD) vaccination system and injected into BALB/c mice that were challenged with S. mansoni cercariae to assess protection and immunogenicity. A total of 39 highly expressed S.mansoni proteins were identified as being of potential interest. Three T cell peptides predicted to bind MHC mouse and human class II were synthesized and formulated for vaccination. SmGSP and SmIKE reduced the number of eggs trapped in the liver by more than 50% in challenged BALB/c mice. The liver of mice vaccinated with either SmGSP or SmTNP had a significantly reduced affected liver surface. Transcriptome-based T cell peptides elicit partial protection and could be candidates for a multiantigen vaccine.

Schistosomiasis is a parasitic disease that affects 143 million people in endemic countries. This work analyzed overexpressed sequences from the cercaria phase to the early schistosomulum phase using bioinformatics tools to predict host interaction and selected proteins for predicting T cell epitopes. The final peptides were chemically synthesized, and their toxicity was evaluated in vitro. Peptides were formulated in the Adjuvant Adaptation (ADAD) vaccination system and injected into BALB/c mice that were challenged with S. mansoni cercariae to assess protection and immunogenicity. A total of 39 highly expressed S. mansoni proteins were identified as being of potential interest. Three T cell peptides predicted to bind MHC mouse and human class II were synthesized and formulated for vaccination. SmGSP and SmIKE reduced the number of eggs trapped in the liver by more than 50% in challenged BALB/c mice. The liver of mice vaccinated with either SmGSP or SmTNP had a significantly reduced affected liver surface. Transcriptome-based T cell peptides elicit partial protection and could be candidates for a multiantigen vaccine.

Background Trichuris trichiura (human whipworm) infects an estimated 477 million individuals worldwide. In addition to T. trichiura, other Trichuris species can cause an uncommon zoonosis and a number of human cases have been reported. The diagnosis of trichuriasis has relied traditionally on microscopy. Recently, there is an effort to use molecular diagnostic methods, mainly qPCR. LAMP technology could be an alternative for qPCR especially in low-income endemic areas. Trichuris muris, the causative agent of trichuriasis in mice, is of great importance as a model for human trichuriasis. Here, we evaluate the diagnostic utility of a new LAMP assay in an active experimental mouse trichuriasis in parallel with parasitological method by using stool and, for the first time, urine samples. Methods Stool and urine samples were collected from mice infected with eggs of T. muris. The dynamics of infection was determined by counting the number of eggs per gram of faeces. A LAMP based on the 18S rRNA gene from T. muris was designed. Sensitivity and specificity of LAMP was tested and compared with PCR. Stool and urine samples were analysed by both LAMP and PCR techniques. Results Trichuris muris eggs were detected for the first time in faeces 35 days post-infection. LAMP resulted specific and no cross-reactions were found when using 18 DNA samples from different parasites. The detection limit of the LAMP assay was 2 pg of T. muris DNA. When testing stool samples by LAMP we obtained positive results on day 35 p.i. and urine samples showed amplification results on day 20 p.i., i.e. 15 days before the onset of T. muris eggs in faeces. Conclusions To the best of our knowledge, we report, for the first time, a novel LAMP assay (Whip-LAMP) for sensitive detection of T. muris DNA in both stool and urine samples in a well-established mice experimental infection model. Considering the advantages of urine in molecular diagnosis in comparison to stool samples, should make us consider the possibility of starting the use urine specimens in molecular diagnosis and for field-based studies of human trichuriasis where possible. Further studies with clinical samples are still needed.

Bovine leukocyte antigens (BoLA) have been used as disease markers and immunological traits in cattle due to their primary role in pathogen recognition by the immune system. A higher MHC allele diversity in a population will allow presenting a broader peptide repertoire. However, loss of overall diversity due to domestication process can decrease a population’s peptide repertoire. Within the context of zebu and taurine cattle populations, BoLA-DRB3 genetic diversity in Spanish Morucha and Colombian Normande cattle was analyzed and an approach to estimate functional diversity was performed. Sequence-based typing was used for identifying 29, 23, 27, and 28 alleles in Spanish Morucha, Nariño-, Boyacá-, and Cundinamarca-Normande cattle, respectively. These breeds had remarkably low heterozygosity levels and the Hardy-Weinberg principle revealed significant heterozygote deficiency. FST and DA genetic distance showed that Colombian Normande populations had greater variability than other phenotypically homogeneous breeds, such as Holstein. It was also found that Spanish Morucha cattle were strongly differentiated from other cattle breeds. Spanish Morucha had greater divergence in the peptide-binding region regarding other cattle breeds. However, peptide-binding region covariation indicated that the potential peptide repertoire seemed equivalent among cattle breeds. Despite the genetic divergence observed, the extent of the potential peptide repertoire in the cattle populations studied appears to be similar and thus their pathogen recognition potential should be equivalent, suggesting that functional diversity might persist in the face of bottlenecks imposed by domestication and breeding. © Copyright © 2020 Bohórquez, Ordoñez, Suárez, Vicente, Vieira, López-Abán, Muro, Ordóñez and Patarroyo.

11 páginas, 1 tablas, 6 figuras, Schistosomiasis is considered a neglected parasitic disease. Around 280,000 people die from it annually, and more than 779 million people are at risk of getting infected. The schistosome species which infect human beings are Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma guineensis, and Schistosoma mekongi. This disease is also of veterinary significance; the most important species being Schistosoma bovis since it causes the disease in around 160 million livestock in Africa and Asia. This work was aimed at designing and developing a genus-specific loop-mediated isothermal amplification (LAMP) method for detecting the most important schistosome species affecting humans and for the species-specific detection of S. bovis. Bioinformatics tools were used for primer design, and the LAMP method was standardised for detecting the ITS-1 region from S. intercalatum, S. haematobium, S. mansoni, S. japonicum, and S. bovis DNA (generic test) and the NADH 1 gene for specifically detecting S. bovis (at different DNA concentrations). Detection limits achieved were 1 pg DNA for S. mansoni, 0.1 pg for S. haematobium, 1 pg for S. intercalatum, and 10 pg for S. bovis. No amplification for S. japonicum DNA was obtained. The LAMP designed for the amplification of S. bovis NADH-1 worked specifically for this species, and no other DNA from other schistosome species included in the study was amplified. Two highly sensitive LAMP methods for detecting different Schistosoma species important for human and veterinary health were standardised. These methods could be very useful for the diagnosis and surveillance of schistosome infections., This work was funded by the Institute of Health Carlos III, ISCIII, Spain (http:// www.isciii.es), grants: RICET RD16/0027/0018 (AMA), PI19/01727 (PFS), PI-0001-2019 Junta de Andalucía (AMA, PFS), European Union cofinancing by FEDER (Fondo Europeo de Desarrollo Regional) “Una manera de hacer Europa” and by the Plan TCUE 2018–2020 European Union cofinancing by FEDER and the Junta de Castilla y León.

Chagas’ disease is a neglected tropical disease caused by Trypanosoma cruzi which is endemic throughout Latin America and is spread by worldwide migration. Diagnosis is currently limited to serological and molecular techniques having variations regarding their sensitivity and specificity. This work was aimed at developing a new sensitive, applicable, and cost-effective molecular diagnosis technique for loop-mediated isothermal amplification-based detection of T. cruzi (Tc-LAMP). The results led to determining a highly homologous satellite repeat region (231 bp) among parasite strains as a molecular marker for diagnosing the disease. Tc-LAMP was performed correctly for detecting parasite DNA (5 fg for the CL Brener strain and 50 fg for the DM28, TcVI, and TcI strains). Assay results proved negative for DNA from 16 helminth species and 7 protozoa, including Leishmania spp. Tc-LAMP based on the highly repeated T. cruzi satellite region is thus proposed as an important alternative for diagnosing T. cruzi infection, overcoming other methods’ limitations such as their analytic capability, speed, and requiring specialized equipment or highly trained personnel. Tc-LAMP could be easily adapted for point-of-care testing in areas having limited resources.

Nowadays, safe and efficacious vaccines represent powerful and cost-effective tools for global health and economic growth. In the veterinary field, these are undoubtedly key tools for improving productivity and fighting zoonoses. However, cases of persistent infections, rapidly evolving pathogens having high variability or emerging/re-emerging pathogens for which no effective vaccines have been developed point out the continuing need for new vaccine alternatives to control outbreaks. Most licensed vaccines have been successfully used for many years now; however, they have intrinsic limitations, such as variable efficacy, adverse effects, and some shortcomings. More effective adjuvants and novel delivery systems may foster real vaccine effectiveness and timely implementation. Emerging vaccine technologies involving nanoparticles such as self-assembling proteins, virus-like particles, liposomes, virosomes, and polymeric nanoparticles offer novel, safe, and high-potential approaches to address many vaccine development-related challenges. Nanotechnology is accelerating the evolution of vaccines because nanomaterials having encapsulation ability and very advantageous properties due to their size and surface area serve as effective vehicles for antigen delivery and immunostimulatory agents. This review discusses the requirements for an effective, broad-coverage-elicited immune response, the main nanoplatforms for producing it, and the latest nanovaccine applications for fighting animal pathogens. Incluye referencias bibliográficas

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi which is endemic throughout Latin America and is spread by worldwide migration. Diagnosis is currently limited to serological and molecular techniques having variations regarding their sensitivity and specificity. This work was aimed at developing a new sensitive, applicable, and cost-effective molecular diagnosis technique for loop-mediated isothermal amplification-based detection of T. cruzi (Tc-LAMP). The results led to determining a highly homologous satellite repeat region (231 bp) among parasite strains as a molecular marker for diagnosing the disease. Tc-LAMP was performed correctly for detecting parasite DNA (5 fg for the CL Brener strain and 50 fg for the DM28, TcVI, and TcI strains). Assay results proved negative for DNA from 16 helminth species and 7 protozoa, including Leishmania spp. Tc-LAMP based on the highly repeated T. cruzi satellite region is thus proposed as an important alternative for diagnosing T. cruzi infection, overcoming other methods' limitations such as their analytic capability, speed, and requiring specialized equipment or highly trained personnel. Tc-LAMP could be easily adapted for point-of-care testing in areas having limited resources.

Schistosomiasis is a significant public health problem in sub-Saharan Africa, China, Southeast Asia, and regions of South and Central America affecting about 189 million people. Kunitz-type serine protease inhibitors have been identified as important players in the interaction of other flatworm parasites with their mammalian hosts. They are involved in host blood coagulation, fibrinolysis, inflammation, and ion channel blocking, all of them critical biological processes, which make them interesting targets to develop a vaccine. Here, we evaluate the protective efficacy of chemically synthesized T- and B-cell peptide epitopes derived from a kunitz protein from Schistosoma mansoni. Putative kunitz-type protease inhibitor proteins were identified in the S. mansoni genome, and their expression was analyzed by RNA-seq. Gene expression analyses showed that the kunitz protein Smp_147730 (Syn. Smp_311670) was dramatically and significantly up-regulated in schistosomula and adult worms when compared to the invading cercariae. T- and B-cell epitopes were predicted using bioinformatics tools, chemically synthesized, and formulated in the Adjuvant Adaptation (ADAD) vaccination system. BALB/c mice were vaccinated and challenged with S. mansoni cercariae. Kunitz peptides were highly protective in vaccinated BALB/c mice showing significant reductions in recovery of adult females (89–91%) and in the numbers of eggs trapped in the livers (77–81%) and guts (57–77%) of mice. Moreover, liver lesions were significantly reduced in vaccinated mice (64–65%) compared to infected control mice. The vaccination regime was well-tolerated with both peptides. We propose the use of these peptides, alone or in combination, as reliable candidates for vaccination against schistosomiasis. © Copyright © 2019 Hernández-Goenaga, López-Abán, Protasio, Vicente Santiago, del Olmo, Vanegas, Fernández-Soto, Patarroyo and Muro.

[EN]Background Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. Methodology/Principal findings The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMPpositive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. Conclusions/Significance The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni.

Strongyloidiasis is a parasitosis that represents a public health problem, in tropical regions. The present study aimed to investigate the anthelmintic effects of several extracts of Argemone mexicana, as well as its main component berberine (Ber) against the third-stage larvae (L3) of Strongyloides venezuelensis in-vitro experiments. Also, the anti-hemolytic activity of the extract, fractions, and Ber were tested in human erythrocytes. A dose-response anthelminthic bioassay demonstrated Ber as the most effective component, followed by methanolic subfraction (Fr3) and finally the crude extract of A. mexicana (Am) showing LC50 response values of 1.6, 19.5, and 92.1 μg/mL, at 96 h respectively. Also, Am, Fr3, and Ber did not produce significant hemolysis against human erythrocytes (p ≤ 0.05). Am and Fr3 showed erythrocyte protection effect capacity at the membrane level (p ≤ 0.05). Furthermore, Ber was found to have an antioxidant activity of 168.18 μg/mL. According to the results, the Fr3 of A. mexicana, and particularly Ber, exhibited potent in-vitro effects against L3 of S. venezuelensis, without hemolytic activity against human erythrocytes and presented good antioxidant capacity. In conclusion, the extracts of A. mexicana and the main component have activity against S. venezuelensis, nevertheless, further studies are required to elucidate the mechanism of action.

Background Crimean-Congo hemorrhagic fever (CCHF) is an emerging infectious disease caused by a Nairovirus. CCHF is a tick-borne disease that is predominantly associated with Hyalomma ticks and have a widespread distribution in Africa, Asia and Europe. CCHF usually presents as a subclinical disease, but in some cases, it may present as a hemorrhagic fever with a high mortality rate. This systematic review of the literature was performed to identify the available evidence on the prevalence of CCHF in the European Region of the World Health Organization, based on seroprevalence (IgG antibodies). Methodology A systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement protocol. PubMed, Embase, and the Web of Science were used for the search (up to January 31, 2019), combining the following MeSH terms: [“Crimean-Congo haemorrhagic fever” OR “Crimean-Congo hemorrhagic fever virus” OR “Congo-Crimea” OR “Crimea-Congo”] AND [“Europe”] AND [“epidemiology” OR “seroprevalence”]. The abstracts were screened. Subsequently, full-text articles were selected and reviewed based on the PICOS (Population-Intervention-Comparison-Outcomes-Study type) criteria by two independent reviewers for inclusion in the final analysis. The data were qualitatively synthesized without quantitative pooling due to the heterogeneity in the study populations and methodologies. Principal findings Thirty articles (9 from western Europe, 18 from central Europe and 3 from eastern Europe) were included in the analysis. All articles were cross-sectional studies (descriptive studies). Conclusions The highest seroprevalence of CCHF is found in central and eastern European countries. Southern and western Europe countries, such as Greece and Spain, have low levels of endemicity, but the spread of the infection, which is associated with climate change, is a possibility that we should keep in mind. Further studies, especially larger seroprevalence studies in humans and animals, are needed to establish the current status of the CCHF epidemiology and to generate standardized guidelines for action in the region., Author summary Crimean Congo hemorrhagic fever (CCHF) is a widespread tick-borne viral disease caused by a Nairovirus of the Nairoviridae family. CCHF-virus (CCHFV) has been considered to be one of the eight priority emergent pathogens for the last 3 years by the World Health Organization (WHO), requiring urgent attention in Research, Development and Innovation (R&D&I) because of its epidemic potential in the near future In this systematic review, we aimed to describe the epidemiological impact of CCHFV (seroprevalence for IgG antibodies and the associated risk factors) in the WHO European Region (WHO/Europe). In this systematic review suggests the following conclusions. i) The highest values of CCHFV seroprevalence are found in Turkey, the Russian Federation, and Kazakhstan. ii) Greece has a high seroprevalence, though only one death associated with CCHFV has been reported. This fact contrasts with the neighboring countries, such as Balkan countries and Turkey, where the rate of severe infections seems to be higher. iii) Extensive studies should be developed in European countries to establish the actual epidemiological situation and to take additional preventive measures for the future.

Objetive: To evaluate the performance of Rapid-Heat LAMPellet assay in field conditions for diagnosis of urogenital schistosomiasis in an endemic area in Cubal, Angola, and to assess the reproducibility in a reference laboratory. Methods: A total of 172 urine samples from school-age children were tested for microhaematuria, microscopic detection of Schistosoma haematobium eggs and LAMP for DNA detection. Urine samples were stored in a basic equipped laboratory. Field-LAMP tests were performed with and without prior DNA extraction from urine samples, and the results were read by turbidity and by colour change. When field procedures were finished, samples were sent to a reference laboratory to be reanalysed by LAMP. Results: A total of 83 of 172 (48.3%) were positive for microhaematuria, 87/172 (50.6%) were microscopy-positive for S. haematobium eggs detection, and 127/172 (73.8%) showed LAMP-positive results for detecting S. haematobium using purified DNA and 109/172 (63.4%) without prior DNA extraction. MacNemar's test showed a statistical significant relation between LAMP results and microscopy-detected S. haematobium infections and microhaematuria (P

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.

The effects of using nitric oxide (NO) donors and inhibitors in experimental strongyloidiasis were showed using, both naïve and dexamethasone immunosuppressed BALB/c mice infected with Strongyloides venezuelensis. Aminoguanidine, an inhibitor of inducible NO synthase and LA419 a NO donor, were administered. Dexamethasone was used to induce immunosuppression. The study in BALB/c mice revealed increases in counts of fecal eggs, larvae in lungs and parasitic females following treatment with aminoguanidine, while mice treated with LA419 had limited egg output with low larval and adult recoveries. Mice immunosuppressed with dexamethasone developed hyperinfection with high long lasting fecal egg emission, high numbers of larvae in lungs and high numbers of parasitic females in the intestine even when the infection had already been cleared in non-immunosuppressed infected controls. Mice treated with dexamethasone and aminoguanidine had the highest egg output and the highest larva and parasitic female recovery showing a severe hyperinfection syndrome. In contrast, treatment with dexamenthasone and LA419 resulted in a controlled hyperinfection syndrome and these mice were able to eliminate the parasite. Therefore, NO modulation appears to be a determinant factor in severe strongyloidiasis and further studies should be conducted to confirm in other experimental models.

Background Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. Methodology/Principal findings A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. Conclusions/Significance We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay., Author summary Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., is a highly prevalent parasitic infection affecting an indigenous Amerindian group, the Chachi, living in rural and remote tropical areas along the Río Cayapas and its tributaries in the north-western coastal rainforest of Ecuador. Very little is known about the clinical course and treatment of this disease, and the only method for diagnosing it is the parasitological microscopic detection of eggs from Amphimerus spp. in patients' stool samples. This method lacks sensitivity, and the morphology of the eggs may be confounded with other liver and intestinal flukes. New diagnostic tools that can improve the sensitivity and specificity for diagnosing Amphimerus spp. infection would be desirable. At present, LAMP technology shows all the characteristics required of a real-time assay with simple operation for potential use in the clinical diagnosis of infectious diseases, particularly in the field conditions in developing countries for most neglected tropical diseases. In this study, we developed and successfully evaluated a LAMP assay for detecting Amphimerus sp. in human stool samples. After further validation, our LAMP assay (LAMPhimerus) could be readily adapted for effective field diagnosis and disease surveillance in amphimeriasis-endemic areas.

Fasciolosis is listed as one of the most important neglected tropical diseases according with the World Health Organization and is also considered as a reemerging disease in the human beings. Despite there are several studies describing the immune response induced by Fasciola hepatica in the mammalian host, investigations aimed at identifying the expression profile of genes involved in inducing hepatic injury are currently scarce. Data presented here belong to a time-course investigation of the gene expression profile in the liver of BALB/c mice infected with F. hepatica metacercariae at 7 and 21 days after experimental infection. The data published here have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE69588, previously published by Rojas-Caraballo et al. (2015) in PLoS One [1].

Background Schistosomiasis remains one of the most common endemic parasitic diseases affecting over 230 million people worlwide. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis and the fresh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. The development of new molecular monitoring assays for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. Our work was focussed on developing and evaluating a new LAMP assay combined with a simple DNA extraction method to detect S. mansoni in experimentally infected snails as a diagnostic tool for field conditions. Methodology/Principal findings A LAMP assay using a set of six primers targeting a sequence of S. mansoni ribosomal intergenic spacer 28S-18S rRNA was designed. The detection limit of the LAMP assay was 0.1 fg of S. mansoni DNA at 63°C for 50 minutes. LAMP was evaluated by examining S. mansoni DNA in B. glabrata snails experimentally exposed to miracidia at different times post-exposure: early prepatent period (before cercarial shedding), light infections (snails exposed to a low number of miracidia) and detection of infected snails in pooled samples (within a group of uninfected snails). DNA for LAMP assays was obtained by using a commercial DNA extraction kit or a simple heat NaOH extraction method. We detected S. mansoni DNA in all groups of snails by using no complicated requirement procedure for DNA obtaining. Conclusions/Significance Our LAMP assay, named Biompha-LAMP, is specific, sensitive, rapid and potentially adaptable as a cost-effective method for screening of intermediate hosts infected with S. mansoni in both individual snails and pooled samples. The assay could be suitable for large-scale field surveys for schistosomes control campaigns in endemic areas., Author Summary Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis worldwide and the snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. Molecular xenomonitoring for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. In our study, we have developed a new simple rapid LAMP assay to detect S. mansoni in Biomphalaria glabrata snails under different situations of infection: early prepatent period, light or low-grade infections and in snails pooled samples. Besides, a simple and rapid method for DNA extraction from snails' tissues was successfully used. This LAMP assay (named Biompha-LAMP) could be potentially useful for large-scale screening in searching infected snails with S. mansoni in field applicable conditions.

Background Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/Principal Findings Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/Significance Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting., Author Summary Human strongyloidiasis, a soil-transmitted infection mainly caused by Strongyloides stercoralis, is one of the most neglected among the so-called neglected tropical diseases (NTDs). The difficult diagnosis lead to an underreporting of infection rates. Strongyloidiasis can easily be misdiagnosed because many infections remain asymptomatic and the lack of sensitivity of the conventional fecal-based techniques for morphologically identification of infective larvae in feces. Although serologic tests are useful, a limitation in standardization to avoid cross-reactions still remains. There is an urgent need to improve more sensitive and specific diagnostic tests, particularly in immunocompromised patients or candidates to immunosuppressive treatments. Several molecular approaches for Strongyloides spp. DNA detection have already been assayed, but they have a very limited use in routine diagnostic, particularly in endemic areas. In addition, all molecular approaches for Strongyloides spp. DNA detection have always been mainly assayed for stool samples and no other more advantageous biological samples, such as urine, have been investigated for molecular purposes. In this study we have developed, for the first time, a molecular assay using LAMP methodology as a simple, sensible and robust method for the detection of S. venezuelensis DNA in a well-established Wistar rats experimental infection in both stool and urine samples. The LAMP assay was also successfully evaluated in patients´ stool samples. Our LAMP assay (Strong-LAMP) is an useful molecular tool in a strongyloidiasis experimental infection model and could be a potential field-friendly diagnostic test in a clinical setting, following further validation.

Background Strongyloidiasis is a parasitic disease widely present in tropical and subtropical areas. Strongyloides stercoralis represents the main species that infects human beings. Ivermectin is the current drug of choice; however, issues related with treatment failure in patients with diabetes or infected with T-lymphotropic virus-1 make the identification of new molecules for alternative treatment a priority. In the present study, the activity of sphingosine-related aminoalcohol and diamine were evaluated against Strongyloides venezuelensis third-stage larva (L3) cultures and experimental infections in mice. Methods The efficacy of each compound against L3 was assessed using both XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and microscopic observation with concentrations ranging from 1 to 350 μM. Cytotoxicity was evaluated using J774.2 macrophage cell line and XTT assay. Lethal concentration 50 (LC50), selectivity index (SI) and structure-activity relationships were established. The activity compounds 4 (2-(ethylamino) hexadecan-1-ol), 6 (2-(butylamino) hexadecan-1-ol), 17 (tert-butyl N-(1-aminododecan-2-yl) carbamate) and 18 (tert-butyl N-(1-aminohexadecan-2-yl) carbamate) were further assessed against experimental S. venezuelensis infections in CD1 mice measuring reductions in the numbers of parthenogenetic females and egg passed in faeces. Mice were infected with 3,000 L3 and treated with 20 mg/kg/day for five days. Results In the screening study of 15 aminoalcohols [lauryl (n = 9); palmityl (n = 13); stearyl (n = 15) and alcohol derivatives], the presence of a palmitol chain was associated with the highest efficacy against L3 (LC50 31.9–39.1 μM). Alkylation of the 2-amino group with medium size fragments as ethyl or n-butyl showed the best larvicidal activity. The dialkylation did not improve efficacy. Aminoalcohols 4 and 6 showed the highest SI (1.5 and 1.6, respectively). With respect to diamine derivative compounds, a chain size of sixteen carbon atoms (palmitoyl chain, n = 13), and the alkylation of the 2-amino group with medium-sized fragments, were associated with the highest lethal activities. The presence of carbamoyl group in diamines 17 and 18 yielded high SI (1.7 and 1.4, respectively). Infected mice treated with aminoalcohol 6 showed reduction in parthenogenetic females (59 %) and eggs in faeces (51 %). Conclusions These results support the potentiality of aminoalcohol and diamine sphingosine-related compounds as suitable prototypes for developing new promising drugs against strongyloidiasis. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1648-5) contains supplementary material, which is available to authorized users.

[Context]: C-6-Geranylated flavonoids possess promising biological activities. These substances could be a source of lead compounds for the development of therapeutics. [Objective]: The study was designed to evaluate their antibacterial and antileishmanial activity. [Materials and methods]: C-6-Geranylated flavanones were tested in micromolar concentrations against promastigote forms of Leishmaniabrazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC values (after 72 h) were calculated and compared with that of miltefosine. Flow cytometry and DNA fragmentation analysis were used the mechanism of the effect. Geranylated flavanones or epigallocatechin gallate were combined with oxacillin, tetracycline, and ciprofloxacin, and the effects of these two-component combinations were evaluated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were established (after 24 h), the synergy was measured by the checkerboard titration technique, and the sums of the fractional inhibitory concentrations (∑FICs) were computed. [Results]: 3′-O-Methyl-5′-O-methyldiplacone and 3′-O-methyldiplacone showed good antileishmanial activities (IC 8–42 μM). 3′-O-Methyl-5′-hydroxydiplacone activates the apoptotic death at leishmanias, the effect of 3′-O-methyl-5′-O-methyldiplacone has another mechanism. The test of the antibacterial activity showed good effects of 3′-O-methyldiplacol and mimulone against MRSA (MIC 2–16 μg/mL), and in six cases, the results showed synergistic effects when combined with oxacillin. Synergistic effects were also found for the combination of epigallocatechin gallate with tetracycline or oxacillin. [Conclusion]: This work demonstrates anti-MRSA and antileishmanial potential of geranylated flavanones and uncovers their promising synergistic activities with antibiotics. In addition, the mechanism of antileishmanial effect is proposed., The work was supported by 93/2012/FaF, 63/2014/FaF, and 94/2013/FaF of UVPS Brno. Authors also thank the Project CIETUS 2010 for support.

Schistosomiasis is one disease produced by helminths, which affect many people in tropical areas. Granuloma formation is the main mechanism involved in the pathogenesis of this disease. Experimental studies have demonstrated angiogenesis (blood vessels formation from pre-existing vessels) in the initial phase of granuloma formation. In the present work, VEGF (vascular endothelial growth factor) levels were analyzed in sera from people diagnosed with different helminthic infections. Patients with schistosomiasis and filariasis had significantly high VEGF levels in compared with healthy people and patients diagnosed with hookworms. In addition, the effects of angiogenesis inhibition using anti-angiogenic factors (endostatin) were evaluated in a schistosomiasis murine model. A lesion decrease was observed in mice infected with Schistosoma mansoni and treated with endostatin. Finally, mechanisms of angiogenesis induction were studied and observed that cercariae antigens stimulated the angiogenic factors by host alveolar macrophages.

Control of Strongyloides stercoralis infection is based on antihelminthic treatment. However, the development of an efficient vaccine is an important goal for more effectively managing this disease. Different Strongyloides spp. antigen preparations have been previously tested but without satisfactory results. In the present study, we evaluated whether the doxycholic acid extract of Strongyloides venezuelensis stage 3 larvae was able to protect CD1 mice against a homologue infection. Moreover, we included saponins from Quillaja saponaria (Qs) and immunomodulatory substances, i.e., Phlebodium pseudoaureum hydroalcoholic extract (PAL) or the amino alcohol AA0029, which has an aliphatic chain of 14 carbon atoms with a hydrophilic amino alcohol head, where the amino group is protected with a butoxycarbonyl group. The DOCSv antigen, together with the adjuvant components, was emulsified in a non-mineral water/oil emulsion. We performed duplicate experiments with each of the 2 immunomodulators. The DOCSv antigen used with Qs+PAL induced high levels of protection in terms of fecal egg count reduction (93.2-99.4%), 86-88% in adult worm recovery, and reduction in swelling of the small intestine. Mice vaccinated with the DOCSv antigen, together with Qs+AA0029, showed a 73.7-97.0% reduction in fecal egg count, with reduction in inflammation of the small intestine. High levels of IgG and IgG2a were observed in mice vaccinated with Qs+AA0029+DOCSv, indicating a Th1 immune response. Also, vaccinated mice recognized bands of 34, 39, 43, 95, and 170 kDa in a Western blot.

Summary This study aims to investigate the role of angiogenic factors in the pathogenesis of experimental strongyloidiasis. Two complementary approaches were used: Firstly, CD1 mice were treated with endostatin, an angiogenesis inhibitor, and infected with Strongyloides venezuelensis. Also, the mechanisms involved in this process were studied. Parasitological examination revealed a significant decrease in egg per gram of faeces, number of collected larvae from lung tissue and number of collected adult females in mice treated with endostatin. Direct mechanisms with diminution of angiogenesis factors and an indirect mechanism with increase of eosinophil perhaps produced their effect. Secondly, the effect of the antigens responsible for stimulation of angiogenic factors [vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2)] from alveolar macrophages and the mechanisms involved in their production were investigated. Alveolar macrophage cells obtained by bronchoalveolar lavage were incubated at different concentrations of somatic and excretory/secretory antigens of S. venezuelensis. Also, mRNA levels of VEGF and FGF2 in macrophage cells were detected by RT-PCR. L3-PBS larvae antigens induced angiogenic factors. The relationship between angiogenesis factors and nitric oxide has been observed using nitric oxide synthase inhibitors.

The newborn larval stage of Trichinella spiralis enters the host striated skeletal muscle cell resulting in the formation of the nurse cell. Vascular Endothelial Growth Factor (VEGF) was detected in cells in the area immediately surrounding the nurse cells. However, no data are available on the antigens involved, the role of other angiogenic factors or the relationship of angiogenesis with Nitric Oxide (NO) production. Using macrophage cell culture we study the effect of different Trichinella L1 antigens from one encapsulated (T. spiralis) and one non-encapsulated (Trichinellapseudospiralis) on the expression of VEGF and basic Fibroblast Growth Factor (FGF2). Also, we investigate the relationship between the production of NO and angiogenic mediators. The results show that encapsulated and non-encapsulated Trichinella species are different in their capacity to stimulate the expression of VEGF and FGF2 from host macrophages. Finally, there is no relationship between angiogenic factors and NO production by T. spiralis antigen.

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.

Currently available candidate vaccines against schistosomiasis elicit only partial protection. In addition, the type of immune response that could lead to the highest level of protection against schistosomes has not yet been described. Thus, efforts should be made in both the identification of novel proteins essential for the parasite cycle and in the modulation of immune responses against these novel candidates through the combined use of immunomodulatory molecules. Several parasites have 14-3-3 proteins, and these proteins are known to play a key role in parasite biology. In the present work, we report the isolation and characterization of a new 14-3-3 gene from Schistosoma bovis and offer new information regarding the genetic structure of the gene. In addition, we have produced the corresponding recombinant protein. Finally, we describe the immune responses elicited by this protein when combined with 4 different immunomodulators in immunized mice.

Schistosoma bovis is a trematode parasite mainly affecting cattle and sheep. Evidences about the arise of drug resistance and the high rates of re-infection of animals in endemic areas have pointed out the need of developing new control tools, e.g., effective vaccines. Schistosomes 14-3-3 proteins have been defined as vaccine candidates against respective infections. We have therefore investigated the protective capacity of the 14-3-3 protein from S. bovis – Sb14ζ – against S. bovis in mice. In addition, we have addressed the influence of the co-administration of four different immunomodulators with the 14-3-3 polypeptide. The values of protection against S. bovis were statistically significant when the Sb14ζ was combined in two independent experiments with the AA0029 (61.0% and 40.31%), AA2829 (49% and 36.3%) and PAL (49% and 40.075%) immunomodulatory molecules. Immune responses from vaccinated animals showed that the highest protection rates do not necessarily match with a dominant Th1-type response.

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41–48 days post-infection. The mice vaccinated with ADAD only with PAL + Fh15 present a survival rate of 40–50% and those vaccinated with ADAD containing PAL + Qs + Fh15 had a survival rate of 50–62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL + Qs + Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.

It is known that excretory/secretory antigens of Fasciola hepatica (ESFh) trigger a Th2-like immune response. Anapsos (A) is an aqueous hydrosoluble extract obtained from the rhizomes of the fern Polypodium leucotomos that has shown immunomodulator effects in some parasitic infections and immunological disorders. In this work we assess the effect of Anapsos and ESFh and Quillaja saponaria extract (Qs) on BALB/c mice and rat alveolar macrophages. Anapsos modulates the response of mice immunized with ESFh, decreasing IgG antibodies in A+ESFh- and A+Qs+ESFh-treated mice and triggering high levels of gammaIFN in spleen cell culture in comparison with ESFh- and Qs + ESFh--treated groups. Moreover, Anapsos showed statistically significant inhibitory effects on the nitrite production by rat alveolar macrophages prestimulated with lipopolysaccharide (LPS) as well as ESFh antigen in comparison with macrophages stimulated only with LPS. The application of ESFh and Anapsos combined avoids this inhibitory effect. Thus, Anapsos modulates the immune response against ESFh in naive mice and on the nitrite production in prestimulated rat aveolar macrophages.

33 p.-6 fig.-1 tab., Strongyloidiasis is widely distributed in the tropical and subtropical areas. Ivermectin is the drug of choice for the treatment. However, the concerns about relying treatment on a single drug make identification of new molecules a priority. Alkylphospholipid analogues, including edelfosine, are a group of synthetic compounds that have shown activity against some parasites. The objective was to assess the in vitro and in vivo activity of edelfosine, miltefosine, perifosine against Strongyloides venezuelensis. Moreover, apoptosis-like mechanism in larvae after treatment was studied. Edelfosine displayed the highest activity and the best selectivity index (LD50 = 49.6 ± 5.4 μM, SI = 1.1) compared to miltefosine or perifosine. Third stage larvae after culture with edelfosine were not able to develop an infection in mice. Treatment of mice with edelfosine showed reduction of 47% in parasitic females allocated in the gut. Moreover, DNA fragmentation was observed by TUNEL staining in larvae treated with edelfosine. These results suggest that edelfosine could be an effective drug against strongyloidiasis, probably through induction of apoptosis-like cell death., This work has been supported by Proyectos Integrados IBSAL (IBY15/00003; Salamanca, Spain; Spanish Ministerio de Economía y Competitividad (SAF2011-30518, SAF2014-59716-R, and RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional of the European Union), and the European Union’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS).

Background Schistosomiasis is a water-borne disease afflicting over 261 million people in many areas of the developing countries with high morbidity and mortality. The control relies mainly on treatment with praziquantel. Fatty acid binding proteins (FABP) have demonstrated high levels of immune-protection against trematode infections. This study reports the immunoprotection induced by cross-reacting Fasciola hepatica FABP, native (nFh12) and recombinantly expressed using two different expression systems Escherichia coli (rFh15) and baculovirus (rFh15b) against Schistosoma mansoni infection. Methods BALB/c mice were vaccinated with native nFh12 or recombinant rFh15 and rFh15 FABP from F. hepatica formulated in adjuvant adaptation (ADAD) system with natural or chemical synthesised immunomodulators (PAL and AA0029) and then challenged with 150 cercariae of S. mansoni. Parasite burden, hepatic lesions and antibody response were studied in vaccination trials. Furthermore differences between rFh15 and rFh15b immunological responses (cytokine production, splenocyte population and antibody levels) were studied. Results Vaccination with nFh12 induced significant reductions in worm burden (83 %), eggs in tissues (82–92 %) and hepatic lesions (85 %) compared to infected controls using PAL. Vaccination with rFh15 showed lower total worm burden (56–64 %), eggs in the liver (21–61 %), eggs in the gut (30–77 %) and hepatic damage (67–69 %) using PAL and AA0029 as immunomodulators. In contrast, mice vaccinated with rFh15b showed only reductions in eggs trapped in the liver and intestine (53 and 60 %, respectively), and hepatic lesions (45 %). We observed a significant rise in TNFα, IL-6, IL-2, IL-4 and high antibody response (IgG, IgG1, IgG2a, IgM and IgE) in mice immunised with either rFh15 or rFh15b. Moreover, mice immunised with rFh15b showed an increase in IFNγ and a decrease in B220 cells compared to untreated mice, and less production of IgG1 and IgM than in mice immunised by rFh15. Conclusions Higher level of protection is obtained by using Fasciola hepatica-derived FABP protein against Schistosoma mansoni infection. Native FABP is more effective than both recombinant systems. It could be due to post-translational modifications or FABP isoform or changes in the recombinant proteins. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1500-y) contains supplementary material, which is available to authorized users.

Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.We found APLs ranked edelfosineperifosineerucylphosphocholinemiltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

Background Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. Methodology/Principal Findings A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). Conclusions/Significance We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas., Author Summary Human schistosomiasis is a disease caused by several species of parasitic worms of the genus Schistosoma that is affecting 200 million people, especially in sub-Saharan Africa. Most people are infected with Schistosoma haematobium, the species that causes urogenital schistosomiasis and also bladder cancer in many chronic infections. The definitive diagnostic test is based on microscopic examination of urine samples to detect parasite eggs. This method has low sensitivity, high day-to-day variability and cannot be carried out in the acute phase of the disease since the parasite has not started yet to lay eggs. New high-throughput diagnostic tools would be desirable, permitting early treatment and preventing the pathology associated with chronic infections. An interesting approach is the loop-mediated isothermal amplification (LAMP) technique because of its simplicity in operation and potential use in clinical diagnosis and surveillance of infectious diseases. In this study, we developed and evaluated a LAMP assay for detection of S. haematobium DNA in patients´ urine samples using heated pellets with no complicated requirement procedure for DNA extraction, namely the Rapid-Heat LAMPellet method. This is a new, easy, rapid and cost-effective LAMP method that should prove useful for mass screening in limited-resource settings in urogenital schistosomiasis-endemic areas.

This is an open access article distributed under the terms of the Creative Commons Attribution License., [Background]: Schistosomiasis is the third most devastating tropical disease worldwide caused by blood flukes of the genus Schistosoma. This parasitic disease is due to immunologic reactions to Schistosoma eggs trapped in tissues. Egg-released antigens stimulate tissue-destructive inflammatory and granulomatous reactions, involving different immune cell populations, including T cells and granulocytes. Granulomas lead to collagen fibers deposition and fibrosis, resulting in organ damage. Praziquantel (PZQ) is the drug of choice for treating all species of schistosomes. However, PZQ kills only adult Schistosoma worms, not immature stages. The inability of PZQ to abort early infection or prevent re-infection, and the lack of prophylactic effect prompt the need for novel drugs and strategies for the prevention of schistosomiasis. [Methodology/Principal Findings]: Using in vitro and in vivo approaches, we have found that the alkylphospholipid analog edelfosine kills schistosomula, and displays anti-inflammatory activity. The combined treatment of PZQ and edelfosine during a few days before and after cercariae infection in a schistosomiasis mouse model, simulating a prophylactic treatment, led to seven major effects: a) killing of Schistosoma parasites at early and late development stages; b) reduction of hepatomegaly; c) granuloma size reduction; d) down-regulation of Th1, Th2 and Th17 responses at late post-infection times, thus inhibiting granuloma formation; e) upregulation of IL-10 at early post-infection times, thus potentiating anti-inflammatory actions; f) down-regulation of IL-10 at late post-infection times, thus favoring resistance to re-infection; g) reduction in the number of blood granulocytes in late post-infection times as compared to infected untreated animals. [Conclusions/Significance]: Taken together, these data suggest that the combined treatment of PZQ and edelfosine promotes a high decrease in granuloma formation, as well as in the cellular immune response that underlies granuloma development, with changes in the cytokine patterns, and may provide a promising and effective strategy for a prophylactic treatment of schistosomiasis., This work was supported by grants from the Junta de Castilla y León (SA342U13, CSI052A11-2, and CSI221A12-2), Real Federación Española de Fútbol-Sociedad Española de Medicina Tropical y Salud Internacional (RFEF-SEMTSI 2013), Spanish Ministerio de Economía y Competitividad (SAF2011-30518, SAF2014-59716-R, and RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), and European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS).

Thirteen aminoalcohols and eight diamines were obtained and tested against Trypanosoma cruzi epimastigotes strains MG, JEM and CL-B5 clone. Some of them were equal or more potent (1.0–6.6 times) than the reference compound nifurtimox. From them, three aminoalcohols and two diamines were selected for amastigotes assays. Compound 5 was as potent as the reference drug nifurtimox against amastigotes of the CL-B5 strain (IC50 = 0.6 µM), with a selectivity index of 54.

SUMMARY We investigated the in vitro effect of total excretory/secretory and somatic antigens from Ascaris suum adults (ESA and SA) and larvae 3 (ESL3 and SL3), and of 10 purified protein fractions from ESA components on rat alveolar macrophage nitric oxide (NO) production. Our results showed that in vitro incubation of macrophages with SA and SL3 antigens of A. suum did not result in NO release from cells, whereas incubation with ESA or ESL3 antigens resulted in the stimulation of NO production by these cells, both in a specific (inhibited by L-NAME and L-canavanine) and dose-dependent manner. In addition, we could demonstrate that a purified ESA fraction consisting of three Coomassie-stained bands of approximately 37, 44 and 46 kDa is involved in the in vitro triggering of NO production by host cells. These three bands were subjected to MALDI-peptide mass fingerprint, showing similarities with phosphoglycerate kinase, elongation factor Tu and enolase molecules, respectively. Future studies will focus on the characterization of these parasite-derived molecules.

Nitric oxide (NO) is one of the most versatile players in the immune system. Most parasites induce inflammation in the host associated with NO production. Here, we compare the in vitro effect of Schistosoma bovis somatic (SbS) and excretory-secretory (SbES) antigens, and excretory-secretory Paragonimus mexicanus adult worm (PmES) molecules on rat alveolar macrophages NO production measured by the Griess method and by RT-PCR. Additionally, we address the divergence of the NO stimulatory/inhibitory effects of these two parasites. Polymyxin B was used to assess possible LPS contamination. In vitro incubation of rat alveolar macrophages with PmES (10 microg/ml) and SbS (50 microg/ml), but not with SbES extracts, resulted in NO production and an increase in iNOS cell mRNA. This production was specific and inhibited by L-NAME and L-canavanine. Different effects were observed when cells were incubated with P. mexicanus and S. bovis antigens.

Fatty acid binding proteins (FABP) from Fasciola hepatica have demonstrated immune cross-protection against schistosomes. The present study was conducted to develop a new formulation of the recombinant FABP rFh15 with the synthetic immunomodulator AA0029 in the adjuvant adaptation (ADAD) vaccination system and to evaluate its ability to induce immune response and protection against the challenge with Schistosoma bovis cercariae. Immunization of BALB/c mice showed high levels of TNFα, IFNγ, interleukin (IL)-2, IL-6, and IL-4 in splenocyte supernatant culture and also high levels of serum specific anti-rFh15 IgG, IgG1, IgG2a IgE and IgM antibodies suggesting a mixed Th1/Th2 immune response. Using this approach, high levels of protection against experimental challenge with S. bovis cercariae were observed in the mouse and hamster models. A marked reduction up to 64% in worm burden, as well as in the number of eggs retained in liver (66%) and intestine (77%) and hepatic lesions (42%), was achieved in vaccinated BALB/c mice. Golden hamsters vaccinated and challenged in similar conditions had reductions in recovered worms (83%), liver eggs (90%), intestine eggs (96%), liver lesions (56%) and worm fecundity (48–80%). These data suggest that formulation of rFh15 in the ADAD vaccination system using the AA0029 immunomodulator could be a good option to drive an effective immunological response against schistosomiasis.